The publications and other materials used herein to illuminate the background of the invention, and in particular, cases to provide additional details respecting the practice, are incorporated by reference.
Photoluminescence and Fluorescence Phenomenon
In traditional fluorescence phenomenon, such as photoluminescence a fluorescent molecule or ion is excited with light. Photons are absorbed by the target molecule or ion and the energy excites the target molecules or ions electrons to a higher energy state. When the excited energy state of the electrons is released, energy is released as a photon emitted by the molecule or ion. Characteristic for this phenomenon is that the energy of photons of the excitation radiation must be greater that of photons of the emission radiation, because part of the absorbed energy is lost in non-radiative processes within molecule or ion. This means that the excitation wavelength needs to be lower than the emission wavelength. This difference between excitation and emission wavelengths is called Stokes-shift.
Traditional fluorescence phenomenon has widely been used in the study of biomolecular interactions. Traditional fluorescence, however, has several limitations regarding use and sensitivity. In practice, because the fluorescence phenomenon is relatively common, the fluorescence originating from sample impurities, sample containers and components of the measurement equipment is a problem in bioassays. This kind of fluorescence is called autofluorescence.
Another problem with traditional fluorescence is small Stokes shift, meaning that excitation and emission wavelengths of the fluorescent compound are close to each other. This makes it difficult to select and limit the measured wavelength range of measurement instruments. The use of fluorescence and the problems related to its use in different bioassays have been described by Soini and Hemmilä, Clin Chem. (1979) 25:353-361, Fluoroimmunoassay: present status and key problems, and Hemmilä, Clin Chem. (1985) 31:359-370, Fluoroimmunoassays and immunofluorometric assays.
The problem of autofluorescence has been tried to be solved by using temporal resolution in fluorescence measurement. Technology called time-resolved fluorometry has been widely described by Soini and Lövgren, CRC Crit Rev Anal Chem (1987) 18: 105-154, Time-resolved fluorescence of lanthanide probes and applications in biotechnology. In time-resolved fluorescence the fluorescent markers are rare-earth metal ions i.e. lanthanide ions or organometallic compounds containing lanthanide ions such as lanthanide chelates or lanthanide cryptates. Lanthanide cryptates have been described in U.S. Pat. No. 6,352,672, Mabile et. al. ja Mathis G, Clin Chem (1993) 39: 1953-1959, Rare earth cryptates and homogeneous fluoroimmunoassays with human sera. Lanthanide chelates have been described by Hemmilä and Mukkala, Crt Rev Clin Lab Sci (2001) Time-resolution in fluorometry technologies, labels and applications in bioanalytical assays. Lanthanide chelate-dyed nanoparticles have been described by Härmä, Soukka and Lövgren, Clin Chem (2001) 47:561-568, Europium nanoparticles and time-resolved fluorescence for ultrasensitive detection of prostate specific antigen, and inorganic lanthanide particles by Beverloo, van Schadewijk, Zijlmans and Tanke, Anal Biochem (1992) 203:326-334, Immunochemical detection of proteins and nucleic acids on filters using small luminescent inorganic crystals as markers. A time-resolved fluorometer for the measurement of longlifetime photo-luminescence of lanthanides has been described in Clin Chem. (1983) 29:65-68, Time-resolved fluorometer for lanthanide chelates—a new generation of nonisotopic immunoassays, by Soini and Kojola.
The lanthanides have a special characteristic feature of long lifetime of fluorescence. This means that if lanthanides are excited with a short duration light pulse with duration of e.g. 1 μs, the fluorescence emission of lanthanides continues for a long duration e.g. 500 μs-1 ms. Generally the autofluorescence originating from sample impurities, sample containers and components of measurement instrument lasts only about 10 ns. When the fluorescent emission is measured after the excitation light pulse has been turned off, e.g. the measurement is started after a 400 μs delay after the excitation light pulse, the background signal caused by autofluorescence has disappeared. This means that the lowest detectable concentration of the target molecule can be very low.
Another benefit of the time-resolved fluorescence is the large Stokes-shift characteristic to lanthanides. For example europium chelates and cryptates have excitation maximum at approximately 340 nm and emission maximum at approximately 616 nm.
Also time-resolved fluorescence has problems. Because lanthanides need high-energy photons (short wavelength) for excitation, the required excitation light source is complex and expensive. Suitable excitation light sources are e.g. Xenon flash lamp and a nitrogen laser. Another problem is that to reduce the long-lifetime autofluorescence and to be transparent to the ultraviolet excitation radiation, the employed optical components of the measurement instrument need to be of very high quality, e.g. all lenses must be pure quartz. A further problem both in traditional and time-resolved fluorescence is the operated wavelength range. Excitation light is in ultraviolet or visible range of the electromagnetic spectra. Absorption of especially ultraviolet light is high in biological sample matrixes. In addition, sample impurities often absorb light at visible range.
A solution for the described problems of traditional fluorescence and time-resolved fluorescence has been presented by Zarling et. al, WO 94/07142. The publication describes the so-called anti-Stokes photoluminescence method. In anti-Stokes photoluminescence the fluorescent marker is a crystal or a molecular structure constructed typically of two different lanthanide ions. These structures have a characteristic feature of being able to absorb two or three low energy photons of higher wavelength at 980 nm to excite one electron to a higher energy-state than the energy of any of the individual photons. As a result the electron is typically excited to a two or three times higher energy state than in traditional fluorescence or time-resolved fluorescence. Relaxation of the excited state can result in emission of one, two or three different principal wavelengths. A key feature is that the emission light is at a lower wavelength than the excitation light. This phenomenon able to produce so called anti-Stokes emission is called up-conversion. It eliminates the autofluorescence problem completely. Different inorganic upconverting phosphor compounds which produce anti-Stokes photoluminescence have been described by Wright, Mufti, Tagg, Webb and Schneider, Proc SPIE—Int Soc Opt Eng (1997) 2985: 248-255, High-sensitivity immunoassay using a novel upconverting phosphor reporter, Zijimans, Bonnet, Burton, Kardos, Vail, Niedbala and Tanke, Anal Chem (1999), 267:30-36, Detection of cell and tissue surface antigens using up-converting phosphors: a new reporter technology, and U.S. Pat. No. 5,891,656, Zarling, Rossi, Peppers, Kane, Faris, Dyer, Ng and Schneider. Methods for excitation of up-converting photoluminescent phosphors using e.g. laser diode excitation at specified wavelength or broad spectrum light source and suitable excitation filter are described by Soukka et al. in WO 2004/086049 and Zarling et al. in U.S. Pat. No. 5,736,410.
Bioassays
The fluorescent labels and fluorescence measurement method described earlier are employed in so called bioassays, which are heterogeneous or homogeneous, requiring separation or separation free, respectively, bioaffinity binding assays or assays for biological effect e.g. enzymatic activity. Bioassays are used to study interaction of biomolecules, e.g. binding of antibodies to target antigen, or progress of enzymatic reaction, e.g. conversion of substrate to product. These kinds of assays are widely described by Price and Newman (eds.), Principles and practice of immunoassay, 1997; Macmillan, London, as well as US 2004/0076948, Pettersson.
Heterogeneous and Separation-based Assays
Traditional bioassays are heterogeneous i.e. separation based assays. In these assays a binder molecule, which is capable to recognize and bind a target molecule present in the sample, is immobilized onto a reaction vessel or another solid-phase. In competitive binding assays, another labelled molecule, which contains a fluorescent compound and able to bind to a molecule immobilized onto a reaction vessel is added to compete with the target molecule present in the sample. The target molecule present in the sample and the labelled molecule containing the fluorescent compound compete in binding to the binding sites of the immobilized molecules on the reaction vessel. After incubation (time for binding reaction to proceed) the reaction vessel is washed to remove both the unbound target molecules and labelled molecules from the vessel. Thereafter, the fluorescence produced by the bound labelled molecules is measured from the reaction vessel. When the sample contains a low concentration of target molecule, the fluorescence signal is high, because the labelled molecules have bound to the immobilized molecules. In case the concentration of target molecules in the sample is high, the binding sites of the immobilized molecules are occupied by target molecules and the labelled molecules have been unable to bind to the immobilized molecules. An assay, which generates an inverse fluorescence signal response to the target molecule concentration in a sample, is called a competitive assay.
Another alternative assay is a non-competitive assay. In a non-competitive assay typically two binder molecules able to both simultaneously recognize a target molecule are employed. One of the molecules is immobilized onto a reaction vessel or another solid-phase. The molecules immobilized on the reaction vessel recognize the target molecules present in a sample and the target molecules will bind onto binding sites of the molecules immobilized onto the reaction vessel. The reaction vessel can now be optionally washed and all the target molecules not bound to binding sites are removed. Thereafter, the other binder molecule labelled with a fluorescent compound is added. This labelled binder molecule recognizes the bound target molecules and will bind to them at a different site than the binder molecules immobilized on the solid-phase. In case no target molecules are bound to the binding sites of the binder molecules immobilized on the reaction vessel, no labelled binder molecules are bound. The reaction vessel can be washed again to separate the bound and the non-bound labelled binder molecule. If the sample contained the target molecule, it was bound to the molecules immobilized on the reaction vessel and thereafter recognized by the labelled molecule. The formed layered complex is called as sandwich-structure. The measured fluorescence signal is relative to the target molecule concentration, and is high if a large amount or high concentration of the target molecule was present in the sample, and low if only a low concentration or small amount or none of the target molecule was present.
In both of the previous assays based on different principles at least a single wash or separation step is required before the fluorescence signal can be measured. This renders the assays complex and requires expensive instrumentation to carry out the assay automatically.
Homogeneous and Separation-free Assays
Fluorescence-phenomenon is associated with a property known as Foerster-type resonance energy transfer or fluorescence resonance energy transfer (FRET). In case, at a short distance, e.g. below 100 nm—preferably below 10 nm, from the fluorescent compound is present another fluorescent compound, which has an excitation wavelength almost equal to emission wavelength of the first fluorescent compound (known as spectral overlapping), the following can occur: the excited state of the first fluorescent compound is not released as radiative emission of a photon, but the excited-state is relaxed by transferring the energy to another fluorescent molecule, which is transferred to an excited state without absorption of a photon. The other fluorescent compound can thereafter release the excited state energy by emitting a photon at a wavelength characteristic to it. The emission wavelength of the other fluorescent compound is higher than the emission wavelength of the first fluorescent compound and some of the energy is lost in the process. The first fluorescent compound is called a donor and the other fluorescent compound an acceptor.
The energy-transfer process between donor and acceptor described above is called either Förster-type resonance energy-transfer or fluorescence resonance energy-transfer (FRET) and it can be utilized to convert the described heterogeneous and separation-based assays to homogeneous and separation-free assays. In homogeneous assays the binder molecules are not immobilized to solid-phase but labelled with the fluorescent compound typically acting as a donor while the second fluorescent compound present in the assay acts as an acceptor. Homogeneous assays based on FRET and lanthanide compounds as donors have been described by Mathis G, Clin Chem (1993) 39: 1953-1959, Rare earth cryptates and homogeneous fluoroimmunoassays with human sera; Blomberg, Hurskainen ja Hemmilä, Clin Chem (1999) 45:855-861, Terbium and rhodamine as labels in a homogeneous time-resolved fluorometric energy-transfer assay of the beta subunit of human chorionic gonadotropin in serum; Meyer, Haase, Hoheisel ja Bohmann WO 2004/096944, and Hemmilä, Hurskainen, Blomberg, Mukkala, Takalo, Kovanen ja Webb WO 98/15830. From publication Latva, Hemmilä, Blomberg ja Hurskainen U.S. Pat. No. 5,998,146 it is known also that spectral overlapping is not strictly required in case of lanthanide ions as donors. Common to all these assays is the use of long-lifetime fluorescent donor compounds, e.g. a lanthanide chelate, in combination with short-lifetime fluorescent acceptor compounds, i.e. so called prompt fluorescent compounds. In this case the fluorescence of the acceptor excited via fluorescence resonance energy transfer (so called sensitized acceptor emission) is also delayed and can be measured with temporal resolution.
Fluorescence Resonance Energy Transfer
Fluorescence resonance energy transfer (FRET) (Förster, T. Intermolecular energy migration and fluorescence. Ann. Physik 1948; 2, 55-75.) (or Förster resonance energy transfer) describes an energy transfer mechanism between two fluorescent molecules or between a fluorescent and a non-luminescent molecule. A fluorescent donor is excited at its specific fluorescence excitation wavelength. By a long-range dipole-dipole coupling mechanism, this excited state is then non-radiatively transferred to a second molecule, the acceptor, which is luminescent and can emit at its specific emission wavelength, or the quencher, which is non-luminescent or luminescent. The donor returns to the electronic ground state. The mechanism is widely employed in biomedical research (reviewed by Selvin P R. The renaissance of fluorescence resonance energy transfer. Nat Struct Biol 2000; 7: 730-734; and Lakowicz, J. Principles of fluorescence spectroscopy, 2nd edition. Plenum Press, New York, 1999).
The FRET efficiency is determined by the distance between the donor and the acceptor, the spectral overlap of the donor emission spectrum and the acceptor absorption spectrum, and the relative orientation of the donor emission dipole moment and the acceptor absorption dipole moment. The FRET efficiency E depends on the donor-to-acceptor distance r with an inverse 6th order law defined byE=1/(1+(r/R0)6)with R0 being the Förster distance of this pair of donor and acceptor at which the energy transfer efficiency is 50%. The Förster distance depends on the overlap integral of the donor emission spectrum with the acceptor absorption spectrum and their mutual molecular orientation.
A process known as time-resolved fluorescence resonance energy transfer or TR-FRET has been developed to increase the signal to noise ratio of sensitized emission of the acceptor. TR-FRET uses lanthanide chelates, cryptates, ions or nanoparticles as donors. TR-FRET has specifically a problem with the measurement of the sensitized emission. As the excitation light source is turned off the original emission of the donor starts to decay exponentially and the energy-transfer further accelerates the decay. In TR-FRET based assays the sensitized emission of the acceptor has an apparent fluorescent lifetime typically much shorter than the lifetime of the donor. Especially, when the donor and acceptor are very close to each others (the FRET process has maximum efficiency) the sensitized acceptor emission can decay very fast, e.g. in a few microseconds instead of tens or hundreds of microseconds preferred for time-resolved detection. Typically, this results in a very weak emission from the acceptor. This has been tried to be solved by Kokko, Sandberg, Lövgren and Soukka [Europium(III)chelate-dyed nanoparticles as donors in a homogeneous proximity-based immunoassay for estradiol, Anal Chim Acta. 503:155-162 (2004)] by using europium chelate—dyed nanoparticles containing tens of thousands of europium chelates to increase the number of donors taking part in the FRET process in a single binding event. Another improvement has been described by Laitala (WO 2005/033709) optimizing the measurement time windows to very short delay. Ideally the sensitized emission of the acceptor should be measured simultaneously with the excitation of the donor, this is however not possible with time-resolved fluorescence.
Competitive Assay
In competitive assay the fluorescent donor compounds coupled to binder molecules are added to a reaction vessel. Target molecules present in sample compete in binding to binding sites of the binder molecules with derivatized analogues of target molecule labelled with fluorescent acceptor compound. The amount of labelled analogues bound to binder molecules has an inverse relationship to target molecules present in the sample. If no target molecules are present in the sample practically all binding sites of the binder molecules are occupied by the labelled analogues. In case of a very high concentration of target molecule present in the sample, the binding sites of the binder molecule are occupied by the target molecules and not available for binding of the labelled analogue. The components of the assay can be added in a single or multiple steps and optionally the assay can be also incubated between the additions.
The FRET-process is only possible when the donor and acceptor are at a short distance from each other, which is true only when the acceptor labelled analogue is bound to the binder molecule conjugated to the donor. Thus the maximum sensitized acceptor emission is produced when the target molecules are not present in the sample or their concentration is very small and the sensitized acceptor emission decreases with increasing concentration of target molecule, which is typical for the competitive assay principle.
Non-competitive Assay
In non-competitive assay two kinds of binder molecules, first labelled with donor and second labelled with acceptor are added to a reaction vessel. Both of the binder molecules are able to bind simultaneous to the target molecule, i.e. they recognize and bind to different site of the target molecule. When a sample is added the labelled binder molecules present in excess recognize the target molecules and complexes containing the first and second binder as well as analyte are formed—this complex wherein the target molecule is between the two binder molecules is called a sandwich-structure. One of the binder molecules is labelled with the donor and the second with the acceptor and the complex contains both the donor and acceptor label within a short distance from each other enabling the FRET-process between the donor and the acceptor. The formation of the complex and thus the measured sensitized acceptor emission is relative to the presence of the target molecule in the sample. In case no target molecule is present in the sample, no complexes are formed. Also in this assay the components of the assay can be added in a single or multiple steps and optionally the assay can be also incubated between the additions.
Problems in Bioassays Based on FRET Process
In a normal FRET assay only the sensitized emission of the acceptor is measured at an acceptor specific emission wavelength. If the sample has absorbance at this wavelength also the emission of the donor can be measured. Assuming that the absorption is equal at the wavelength of the donor emission and the wavelength of sensitized acceptor emission, the ratio between the donor and the sensitized acceptor emission is independent on the absorption. Donor concentration in all samples is equal and thus, if only a small amount of donors participate in the FRET process, the donor emission can be considered as an internal standard. Alternatively, the donor emission measured from the sample compared to donor emission from a reference sample with no absorbance can be used as a correction factor to estimate the true sensitized emission in the actual sample. These methods have been described in U.S. Pat. Nos. 5,527,684 and 6,352,672 by Mabile et. al. They correct also the variation of absorption at wavelength of excitation.
Another problem in the FRET-based bioassays is radiative energy-transfer. The normal radiative emission of the donor can be reabsorbed by acceptor molecules not at short distance from the donor (unable to participate in the FRET-process) according to Beer-Lambert law and these acceptors generate delayed emission at acceptor specific wavelength. This emission is called radiative energy transfer or non-proximity energy transfer and causes unwanted background to the measured signal. As the emission wavelength is at the acceptor maximum this cannot be spectrally eliminated. However, the apparent decay time of the radiative energy transfer is typically equal to the lifetime of the donor in absence of the acceptor, and thus longer than the apparent lifetime of the sensitized acceptor emission due to FRET. The excited state of the long-lifetime luminescent donor participating in the FRET process is released by two competing processes—the radiative decay typical to the donor and the FRET process, whereof the first is typically constant but the second decay is strongly dependent on the distance between the donor and the acceptor. This results in that the observed apparent radiative decay of the donor participating in FRET is equal or faster than the decay of the donor alone but slower than the observed apparent decay of the sensitized acceptor emission. Thus, effective energy transfer to the acceptor releases the donor excitation state faster than its own characteristic long-lifetime emission. The greater the FRET efficiency is the shorter the observed apparent decay of the donor will be.
Radiative energy transfer process life-time can still be considered practically equal to the donors own characteristic long-lifetime emission because typically most of the signal due to radiative energy transfer is generated by donors not participating in energy transfer—this is especially true when the binder labelled with the donor is used in a large excess to the target molecule, which is typically in a non-competitive assay.
Perhaps the greatest problem of using time-resolved fluorescence in FRET type assays is due to the fact that after the excitation light pulse has been turned of the emission drops exponentially. Even though this creates better signal to noise ratios than in direct fluorescent measurement, the temporal resolution drops the fluorescent signal of the acceptor dramatically.
Known Use of the FRET Process
The FRET process was first used in homogenous assays by Ullman [Ullman, E. F., Schwarzberg, M., and Rubenstein, K. E. (1976) Fluorescent excitation transfer immunoassay. A general method for determination of antigens. J. Biol. Chem. 251, 4172-4178]. He used traditional (short-lifetime, i.e. prompt fluorescent) fluorescent molecules as donors and acceptors. FRET process has been used also with time-resolved fluorescent labels. The described methods, however, suffer the problems associated with traditional fluorescence and time-resolved fluorescence. Anti-Stokes photoluminescence labels as donors in the FRET process are described by Soukka, Härmä and Lövgren in WO 2004/086049. This publication also describes the unwanted background signal originating from the radiative energy transfer. Anti-Stokes FRET is also described in detail in Immunoassay designs and potential of particulate photoluminescent lanthanide reporters, Soukka, 2003; Gillot Oy, Turku, Suomi; ISBN 951-29-2393-9. The publication also lists long-lifetime anti-Stokes photoluminescent donors, short-lived acceptors and a method of measurement.
From the publications Laitala WO 2005/033709 and Ming, Rev Sci Instr (1999), 70:3877-3881. An Improved instrument for measuring time-resolved lanthanide emission and resonance energy transfer; it is known that when a traditional long-lifetime (down-converting) photoluminescent donor and a short lived acceptor is used, optimal measurement is performed as soon as possible after the excitation light pulse has been turned off.